The long term of the proposed studies are to explore the role of oxidized derivatives of cholesterol (oxysterols) in the regulation of cellular cholesterol metabolism and the pathogenesis of atherosclerosis. The hypothesis to be tested in these studies is that oxysterols, which enter the circulation as contaminants of ingested cholesterol-containing foods or as a result of lipoprotein oxidation (in vitro in these studies but which may occur in vivo), can alter cellular cholesterol metabolism. The limited data which are available on the effects of trace levels of oxysterols indicate the their potential effect on cellular cholesterol metabolism may be significant and important. Cholesterol and oxysterols will be monitored in these studies by mass and by radioactivity, using high pressure liquid chromatography (HPLC) with detection of ultraviolet absorption (for mass) and flow-through liquid scintillation counting (for [3H] or [14C]). Rabbits will be fed a mildly hypercholesterolemic diet with or without trace levels of an oxysterol for one month then dosed with radiolabelled cholesterol and/or oxysterol prior to sacrifice. The sterol composition of lipoproteins from rabbits fed the mildly hypercholesterolemic diet contaminated with trace amounts of individual oxysterol species will be compared to that in lipoproteins from rabbits fed the purified cholesterol diet. The distribution of dietary oxysterols relative to cholesterol in certain tissues (liver, heart, kidneys, red blood cells) during the cholesterol/oxysterol feeding. The nature and amount of oxysterols generated during cell-mediated lipoprotein oxidation in vitro (human low density lipoprotein and rabbit lipoproteins including beta very low density lipoprotein) will be monitored. The uptake of lipoprotein-borne oxysterols, derived from dietary sources and lipoprotein oxidation, relative to that of cholesterol and the subsequent cellular fate of these sterols will be examined in vitro in a variety of cell lines representative for the cell types important in cholesterol metabolism. The effect of oxysterols taken up from these lipoproteins on the activity of cellular enzymes involved in cholesterol metabolism will also be examined. It is hoped that these studies will further understanding of the effects of physiologically relevant levels of oxysterols on cellular cholesterol metabolism and will provide insight into the potential role in the development of atherosclerosis.